Transcriptional amplification with two rAAVs using tetracycline-controlled gene induction
Cell-type specific expression of genetically encoded indicators or optogenetic probes is often hampered by the use of promoters that are specific but drive expression only weakly. In a recent paper of the Wang Lab at Princeton University, the authors describe a means of introducing a calcium indicator using the TET promoter system in the cerebellar cortex to boost expression by about ten-fold, as determined by quantitative determination of intracellar concentration. Kuhn et al. show specific labeling of Purkinje cells and all interneuron types. Together with a previous paper of the Wang Lab where glial cells were targeted, now nearly all cell types of the cerebellar cortex can be selectively labeled. Additionally, Kuhn et al. overcome cell toxicity associated with rAAV injection and/or local GECI overexpression by systemic pre-injection of hyperosmotic D-mannitol, doubling the time window for functional imaging.
Interneurons of the cerebellar cortex are labeled using an adeno-associated viral vector delivering the gene of a genetically encoded calcium indicator under control of the human synapsin promoter. The image shows two types of cerebellar interneurons, Lugaro cells (left) and a large Golgi cell (right). The false-color code indicates depth in micrometers below the brain surface. The cells were imaged in vivo with two-photon microscopy and could be functionally imaged for many days. (From Kuhn et al. 2012)
Purkinje cells can be labeled by using an adeno-associated viral vector system expressing a genetically encoded calcium indicator under the TET promoter activated by the transactivator under human synapsin promoter control. The TET system boosts expression about 10-fold compared to expression under the synapsin promoter only. Purkinje cells have a pear-shaped soma and a fan-like dendritic tree. They are completely separated from one another and neatly aligned, as can be seen in the parasagittal (left) and medial-lateral (right) reconstruction. The brain surface is at the top of the images. The cells were imaged in vivo using two-photon microscopy. (From Kuhn et al. 2012)
Functional calcium imaging of Purkinje cell dendrites in awake mice after boosting of expression with the TET promoter. Each stripe (left top) is a dendrite of a Purkinje cell (compare Figure 2) and is color coded (left bottom). The corresponding functional signals were simultaneously recorded in an awake mouse, as shown on the right. Each upstroke in the traces, indicated by a red dot, represents a dendritic complex spike. (From Kuhn et al. 2012)
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